Cannabinoid Inhibition of Adenylate Cyclase-mediated Signal Transduction and Interleukin 2 (IL-2) Expression in the Murine T-cell Line, EL4.IL-2

doi:10.1074/jbc.271.22.13175

Cannabinoid Inhibition of Adenylate Cyclase-mediated Signal Transduction and Interleukin 2 (IL-2) Expression in the Murine T-cell Line, EL4.IL-2*

From the Department of Pharmacology & Toxicology and Department of Pathology, Michigan State University, East Lansing, Michigan 48824-1317

^§ To whom correspondence should be addressed:
Dept. of Pharmacology and Toxicology, B-330 Life Sciences Bldg., Michigan State University, East Lansing, MI 48824.
Tel.: 517-353-3786; Fax: 517-353-8915.

^‡ Present address: Dept. of Life Science, Korea Advanced Institute of Science and Technology, Taejon, Korea.

Abstract

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Δ⁹-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Δ⁹-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

Footnotes

↵* This work was supported by funds from National Institute on Drug Abuse Grants DA07908 and DA09171. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CB

cannabinoid receptor

Δ⁹-THC

Δ-9-tetrahydrocannabinol

G protein

guanine nucleotide-binding protein

PMA

phorbol 12-myristate 13-acetate

IL-2

interleukin 2

bp

base pair(s)

kb

kilobase(s)

rc

recombinant

RT-PCR

reverse transcription-polymerase chain reaction

IS

internal standard

DTT

dithiothreitol

PKA

protein kinase A

CRE

cAMP response element

ELISA

enzyme-linked immunosorbent assay

CREB

cAMP response element-binding protein

ATF

activating transcription factor

PKI

protein kinase A inhibitor.
↵² A. R. Schatz, M. Lee, R. Condie, and N. E. Kaminski, submitted for publication.
↵³ R. B. Crawford, S. G. Hwang, and N. E. Kaminski, unpublished observation.
- Received September 20, 1995.
- Revision received March 15, 1996.